Abstract Intestinal injury and inflammation in Clostridium difficile infection (CDI), an increasing cause of morbidity, mortality and financial cost in the US, is mediated by two large clostridial toxins: TcdA & TcdB. Although antibodies against each toxin have been shown to be associated with protection, it is unclear what types of antibodies and their epitopes are responsible for effective immunity against CDI in patients. The goal of this project is to define the characteristics of a protective humoral immune response in CDI and the major neutralizing TcdA & TcdB epitopes. The hypothesis is that host antibodies directed against major neutralizing epitopes in TcdA & TcdB confer protection against CDI. To test this hypothesis, we will first validate our preliminary finding on neutralizing anti-TcdA/TcdB responses that correlate protection against severe CDI will also correlate protection against recurrence (Aim 1); subsequently we will characterize C. difficile toxin-specific B cell responses at clonal level and correlate these with CDI disease progression and recurrence (Aim 2); and finally we will clone neutralizing antibodies from individual B cells to obtain a representative panel of human protective monoclonal antibodies against the two toxins and characterize their binding epitopes and reactivity to a variety of toxins produced by major C. difficile endemic and outbreak strains (Aim 3). The completion of these specific aims can lead to the discovery of antibody biomarkers that predict outcomes of the most significant clinical issues in CDI management, including CDI occurrence, severity, and recurrence. Identification and characterization of protective antibodies may also facilitate the development of novel passive immunotherapy and vaccine approaches.